US20240016978A1 - Efficient biphasic calcium phosphate coating method - Google Patents

Efficient biphasic calcium phosphate coating method Download PDF

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US20240016978A1
US20240016978A1 US18/249,044 US202118249044A US2024016978A1 US 20240016978 A1 US20240016978 A1 US 20240016978A1 US 202118249044 A US202118249044 A US 202118249044A US 2024016978 A1 US2024016978 A1 US 2024016978A1
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Gang Wu
Liyong Wu
Yunyu LU
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Hangzhou Huibo Science And Technology Co Ltd
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28Materials for coating prostheses
    • A61L27/30Inorganic materials
    • A61L27/32Phosphorus-containing materials, e.g. apatite
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02Inorganic materials
    • A61L27/04Metals or alloys
    • A61L27/06Titanium or titanium alloys
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28Materials for coating prostheses
    • A61L27/30Inorganic materials
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/42Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having an inorganic matrix
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54Biologically active materials, e.g. therapeutic substances
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/12Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/10Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/112Phosphorus-containing compounds, e.g. phosphates, phosphonates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412Tissue-regenerating or healing or proliferative agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412Tissue-regenerating or healing or proliferative agents
    • A61L2300/414Growth factors
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602Type of release, e.g. controlled, sustained, slow
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2420/00Materials or methods for coatings medical devices
    • A61L2420/02Methods for coating medical devices
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00Materials or treatment for tissue regeneration
    • A61L2430/02Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • the present disclosure belongs to the field of preparation of biological coating materials, in particular to a high-efficiency biphasic calcium phosphate coating method with a high yield and a protein encapsulation efficiency.
  • Titanium and titanium alloys have been used in the fields of human hard tissue implants such as artificial joints, fracture fixators, etc. due to excellent characteristics in mechanical performances, biocompatibility, and corrosion resistance.
  • the titanium and titanium alloys are directly implanted into a human body, since the titanium and an oxide film naturally formed on a surface are biologically inert, they are difficult to form a connection with a tissue and promote a formation of a new bone on the surface in an early stage of implantation, thereby showing problems of a low binding force with bones, a poor bioactivity, a long healing time, etc.
  • it is effective to perform a biologically active coating treatment on the surface thereof.
  • a calcium phosphate (CaP) coating also known as a biphasic calcium phosphate coating, is a most widely used surface modification method to promote an osteointegration ability and an osteoconductivity of an endosteal implant and a bone defect filling material due to an osteoid composition, a good biocompatibility, and an ability to regulate various osteogenic processes.
  • the physical deposition technology comprises various thermal processes such as a vacuum plasma spraying and a suspension plasma spraying.
  • a main limitation of the physical deposition technology is that a non-physiological coating condition (>1,000° C.) makes it impossible to incorporate a biologically active substance capable of promoting osteogenesis. Therefore, it is a new trend to develop a new wet chemical deposition technology to prepare a bioactive CaP coating in the field of biomaterial research.
  • a biomimetic coating technology was originally proposed in the 90s of the 20th century.
  • a biomaterial is immersed in a simulated body fluid (SBF) with an ionic concentration, a pH value, and a temperature close to that of human body plasma, and thus a dense, uniform, and typically low-crystallinity apatite can be formed.
  • SBF simulated body fluid
  • This physiological condition enables a large amount of the biologically active substances to co-precipitate with a biomimetic coating and retains their biological activity.
  • an implementation of an initial biomimetic coating process is limited due to its time consuming (typically 14 days) and dependence on a functional group nucleation of the biomaterial.
  • Taset et al. successfully accelerated a coating process significantly to several hours by increasing an SBF concentration by 10 times (10 ⁇ SBF).
  • a problem of SBF deposition depending on biomaterial functional groups remains unsolved.
  • biphasic biomimetic coating method 1) an amorphous CaP base layer was formed by incubation in a 5 ⁇ SBF solution followed by incubation in a supersaturated calcium phosphate solution (SCPS) to form a CaP crystalline layer.
  • SCPS supersaturated calcium phosphate solution
  • This biphasic biomimetic method can form a CaP crystal coating within 3 days.
  • such coating can be used in a variety of biomaterials with different three-dimensional geometries, surface structures, and surface chemical properties, such as metals (titanium alloys), inorganic materials (TCP and Bio Oss), and polymeric materials (naturally derived collagen and synthetic poly lactic-co-glycolic acid).
  • the wide applicability of such biomimetic coating is largely attributable to the amorphous CaP base layer, which can form a mechanical interlocking mechanism rather than a chemical reaction with reactive chemical groups.
  • the amorphous CaP layer is necessary for growth of the subsequent CaP crystalline layer.
  • the CaP crystalline layer can be used for coprecipitation, loading, and slow release of various biologically active substances, in particular an osteoinductive growth factor, a bone morphogenetic protein 2 (BMP-2).
  • BMP-2 bone morphogenetic protein 2
  • An osteogenic induction efficiency of the BMP-2 added to the coating is significantly improved compared to the BMP-2 adsorbed by a surface, which may be related to a controlled release characteristic of the coating.
  • the present disclosure provides an efficient biphasic calcium phosphate coating method with high yield and protein encapsulation efficiency.
  • a high-concentration (4.5 ⁇ ) supersaturated calcium phosphate solution (SCPS) is used for coating and a thicker continuous coating is formed on a titanium disc.
  • SCPS supersaturated calcium phosphate solution
  • the coating contains dicalcium phosphate dihydrate and hydroxyapatite.
  • the coating has an obviously improved encapsulation efficiency of a fluorescently-labeled model protein FITC-BSA and releases less Ca 2+ under an acidic condition.
  • the improved coating process provided by the present disclosure can obtain an osteogenic implant material very expected to be used in the biomedical field with a thicker coating and a higher encapsulation efficiency of biologically active substances by a less SCPS solution volume and a shorter coating time.
  • the present disclosure provides a biphasic calcium phosphate coating, wherein the coating contains dicalcium phosphate dihydrate (DCPD) and hydroxyapatite.
  • DCPD dicalcium phosphate dihydrate
  • the biphasic calcium phosphate coating provided by the present disclosure is a complex of DCPD and hydroxyapatite.
  • DCPA dicalcium phosphate anhydrous
  • OCP octacalcium phosphate
  • pHAp precipitated hydroxyapatite
  • the DCPD can be used in the preparation of a calcium phosphate (CaP) bone cement. Studies find that the DCPD can be converted to hydroxyapatite in vivo and can be degraded and replaced by a bone.
  • the coating containing the DCPD and hydroxyapatite provided by the present disclosure has a firmer structure, deposits more bionic calcium phosphate (CaP), can carry more active proteins, has an acid resistance for resisting osteoclastic resorption, releases less Ca 2+ under an acidic condition, is more slowly degraded in vivo, and has an obviously better performance than a coating only containing the hydroxyapatite.
  • CaP bionic calcium phosphate
  • the present disclosure provides a preparation method, comprising the following steps:
  • the preparation of the biphasic calcium phosphate coating mainly uses the SCPS with a higher concentration to perform coating, thereby realizing an improvement of a coating effect.
  • step (2) the SCPS at a concentration of 1 time or more is 4.5 ⁇ SCPS.
  • the 5 ⁇ SBF in the present disclosure refers to the SBF with a concentration of 5 times; the 4.5 ⁇ SCPS refers to the SCPS with a concentration of 4.5 times; and the 1 ⁇ SCPS refers to the SCPS with a concentration of 1 time.
  • the inventors have surprisingly found through a large amount of researches and experiments that the efficient biphasic calcium phosphate coating with high yield and protein encapsulation efficiency can be realized by coating with the SCPS with a higher concentration, especially with the 4.5 ⁇ SCPS.
  • the coating is a complex of the DCPD and hydroxyapatite, and firmer, releases less Ca 2+ under an acidic condition, degrades more slowly in vivo, and has a coating effect obviously superior to that of the 1 ⁇ SCPS.
  • inorganic ion compositions of the 5 ⁇ SBF, the 1 ⁇ SCPS, and the 4.5 ⁇ SCPS are shown in the following table:
  • step (2) the base layer is taken out, dried, immersed in the 4.5 ⁇ SCPS, and incubated at room temperature for 5 h or more.
  • step (1) the titanium disc is immersed into the 5 ⁇ SBF and incubated at 37° C. for 24 h to prepare the CaP base layer.
  • the CaP base layer is an amorphous CaP, can be used for coprecipitation, loading, and slow release of various biologically active substances, is necessary for growth of the subsequent CaP crystalline layer, and can provide a crystallizing nuclei for formation of a subsequent crystal.
  • the titanium disc has a length and a width of 4 mm and a thickness of 1 mm.
  • step (2) the 4.5 ⁇ SCPS has a volume of 0.1-6 ml.
  • the volume of the 4.5 ⁇ SCPS is in two different relationships with a coating thickness.
  • the coating thickness increases rapidly.
  • the volume of the 4.5 ⁇ SCPS increases to 1 ml or more, especially when the solution volume is relatively high (2-6 ml), the coating thickness increases relatively slow.
  • the coating with a clear crystal structure can be obtained, and the amorphous CaP cannot be observed by a field emission scanning electron microscope.
  • 0.3 ml of the 4.5 ⁇ SCPS can form a continuous coating and 1 ml of the 4.5 ⁇ SCPS can already form a very thick coating. It can be seen that the coating with a higher performance can be achieved with a less volume with the 4.5 ⁇ SCPS.
  • the present disclosure provides use of 4.5 ⁇ SCPS in preparing a biphasic calcium phosphate coating, wherein the biphasic calcium phosphate coating contains dicalcium phosphate dihydrate and hydroxyapatite.
  • the present disclosure provides use of 4.5 ⁇ SCPS in improving one or more of a yield, a protein encapsulation efficiency, and a coating thickness of a biphasic calcium phosphate coating.
  • FESEM field emission scanning electron microscope
  • FIG. 3 A- 3 B show results of detected pH values of solutions and Ca 2+ levels in solutions in example 6, wherein FIG. 3 A shows the pH values in 1 ⁇ SCPS and 4.5 ⁇ SCPS during a coating preparation; and FIG. 3 B shows changes of Ca 2+ along with time; and *, compared with a previous time point; p ⁇ 0.05;
  • FIG. 4 A- 4 B show EDS analysis results of 1 ⁇ SCPS and 4.5 ⁇ SCPS coatings in example 7, wherein FIG. 4 A is an EDS energy spectrum analysis of surface CaP crystalline coatings of titanium discs containing amorphous CaP coatings after incubation in the 1 ⁇ SCPS or 4.5 ⁇ SCPS for 48 h; and FIG. 4 B is a calcium-phosphorus ratio calculated by analyzing data using EDS;
  • FIG. 5 A- 5 B show attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) analysis spectrums in example 7 to analyze chemical compositions of CaP coatings after incubation of titanium discs containing amorphous CaP coatings in 1 ⁇ SCPS FIG. 5 A or 4.5 ⁇ SCPS FIG. 5 B for 5 h, 24 h, and 48 h;
  • ATR-FTIR attenuated total reflection-fourier transform infrared spectroscopy
  • FIG. 6 A- 6 B show x-ray diffraction (XRD) analysis patterns in example 7 analyzing phase compositions of CaP coatings after incubation of titanium discs containing amorphous CaP coatings in 1 ⁇ SCPS FIG. 6 A or 4.5 ⁇ SCPS FIG. 6 B for 5 h, 24 h, and 48 h;
  • XRD x-ray diffraction
  • FIG. 8 shows an encapsulation efficiency of fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) as a model protein in coatings of titanium discs in 1 ⁇ SCPS or 4.5 ⁇ SCPS groups in example 9, where, ***, p ⁇ 0.001.
  • FITC-BSA fluorescein isothiocyanate-labeled bovine serum albumin
  • the macroscopic crystalline coating can be repeated prepared on the titanium disc of the amorphous CaP coating using the 4.5 ⁇ SCPS.
  • the effect was obviously better than that of the 5 ⁇ SCPS. Therefore, the 5 ⁇ SCPS failed to form a crystalline coating and the 4.5 ⁇ SCPS was preferably recommended for coating.
  • a 1 ⁇ SCPS coating in the present example was prepared by using the coating prepared by the method provided in example 1, wherein volumes of 1 ⁇ SCPS for immersing a titanium disc were 0.1, 0.3, 1, 2, 4, and 6 ml respectively.
  • a 4.5 ⁇ SCPS coating was prepared by using the coating prepared by the method provided in example 2, wherein volumes of 4.5 ⁇ SCPS for immersing a titanium disc were 0.1, 0.3, 1, 2, 4, and 6 ml respectively.
  • Thicknesses of the 1 ⁇ SCPS coating and the 4.5 ⁇ SCPS coating were compared.
  • a volume dependence of a CaP crystalline layer formed on the titanium disc with a length and a width of 4 mm respectively and a thickness of 1 mm was examined.
  • a detection method of the coating thickness was as follows: a titanium disc was embedded with methyl methacrylate, and a 200- ⁇ m thick specimen was cut perpendicularly along a cross section of the titanium disc, stuck on an organic glass holder, ground to 80 further dyed with a 0.1% basic fuchsin, and observed with a cold light source (KL 2500 LCD; Carl Zeiss) of a stereo microscope (Stemi SV6; Carl Zeiss, Jena, Germany) and photographed.
  • KL 2500 LCD Carl Zeiss
  • FIGS. 1 A- 1 B It can be seen in FIGS. 1 A- 1 B that in groups 1 ⁇ SCPS and 4.5 ⁇ SCPS, a higher SCPS volume produced a higher coating thickness. 0.3 mL of the 4.5 ⁇ SCPS formed a continuous coating, while 0.3 mL of the 1 ⁇ SCPS formed a thin and discontinuous coating ( FIG. 1 A ). Coating is a method for promoting osteointegration and also a method for carrying proteins. The discontinuous coating represents an incomplete coating, promotes a reduction of an osteointegration capacity, and carries a low amount of proteins.
  • the coating thickness of the group can be increased proportionately by increasing the volume of the 1 ⁇ SCPS.
  • the volume of the 4.5 ⁇ SCPS was in two different relationships with the coating thickness. When the volume of the 4.5 ⁇ SCPS was in a range of 0.1-1 mL, the coating thickness increased rapidly. When the solution volume was relatively high (2-6 mL), the coating thickness increased relatively slowly.
  • a 1 ⁇ SCPS coating in the present example was prepared by using the coating prepared by the method provided in example 1, wherein volumes of 1 ⁇ SCPS for immersing a titanium disc were 0.1, 0.3, 1, 2, 4, and 6 ml respectively, and incubation time was 48 h.
  • a 4.5 ⁇ SCPS coating was prepared by using the coating prepared by the method provided in example 2, wherein volumes of 4.5 ⁇ SCPS for immersing a titanium disc were 0.1, 0.3, 1, 2, 4, and 6 ml respectively, and incubation time was 48 h.
  • the amorphous CaP degraded quickly, had a small thickness of about 100-300 nm, and carried very less proteins.
  • the amorphous CaP in the present application primarily served as a precursor to provide a crystallizing nuclei for formation of a subsequent crystal.
  • a coating thickness (73 ⁇ m) of 1.5 mL of 4.5 ⁇ SCPS was similar to a coating thickness produced by 6 mL of 1 ⁇ SCPS on a single titanium disc (with a length and width of 4 mm and a thickness of 1 mm). Therefore, a 1 ⁇ SCPS coating in the present example was prepared by using the coating prepared by the method provided in example 1, wherein a volume of 1 ⁇ SCPS for immersing a titanium disc was 6 ml.
  • a 4.5 ⁇ SCPS coating was prepared by using the coating prepared by the method provided in example 2, wherein a volume of 4.5 ⁇ SCPS for immersing a titanium disc was 1.5 ml.
  • pH values of the solution and Ca 2+ levels in the solution at different time points were measured respectively at different time points.
  • the Ca 2+ content in the solution was measured using an atomic absorption spectrometer (AAnalyst 100, PerkinElmer, Foster City, CA, USA). Data were presented as mean and standard deviation. Data on changes of the pH values and Ca 2+ levels were compared using a one-way analysis of variance (ANOVA). A Post-hoc comparison was performed using a Bonferroni correction and a significance level was p ⁇ 0.05.
  • FIGS. 3 A and 3 B The results were shown in FIGS. 3 A and 3 B , wherein FIG. 3 A showed the pH values of the 1 ⁇ SCPS and 4.5 ⁇ SCPS during a coating preparation process.
  • FIG. 3 B showed changes of Ca 2+ along with time; and *, compared with a previous time point; p ⁇ 0.05;
  • the Ca 2+ concentrations of the 1 ⁇ SCPS group decreased significantly within 5 h from 4.0 mM to 3.2 mM (p ⁇ 0.05), and gradually to 2.6 mM. About 35% of the Ca 2+ was consumed at an end of a coating process. With regard to 4.5 ⁇ SCPS, the Ca 2+ concentration decreased significantly (p ⁇ 0.05) after 5 h and 24 h of incubation, but subsequently remained stable at about 13 mM, and did not further decrease. About 30% of the Ca 2+ was consumed at an end of the coating preparation process.
  • a 1 ⁇ SCPS coating in the present example was prepared by using the coating prepared by the method provided in example 1, wherein a volume of 1 ⁇ SCPS for immersing a titanium disc was 6 ml.
  • a 4.5 ⁇ SCPS coating was prepared by using the coating prepared by the method provided in example 2, wherein a volume of 4.5 ⁇ SCPS for immersing a titanium disc was 1.5 ml.
  • a calcium-phosphorus ratio was calculated by analyzing a relative density of calcium and phosphorus in the coating through EDS.
  • a phase composition was detected by XRD (X'Pert PRO; PANalytical, Malvern, UK) and chemical groups were analyzed by ATR-FTIR.
  • a scanning range of an XRD analysis was 2.00°-80.00°, a scanning speed was 2°/min, and a scanning interval was 0.02°.
  • FTIR Anatar 360 Nicolet spectrometer; Thermo Fisher Scientific, Waltham, MA, USA
  • detected by ATR-FTIR was a transmission spectrum with the wavenumber of 4,000 to 400 cm ⁇ 1 .
  • FIGS. 4 A and 4 B The EDS analysis results were shown in FIGS. 4 A and 4 B , wherein FIG. 4 A was an EDS energy spectrum analysis of surface CaP crystalline coatings of titanium discs containing amorphous CaP coatings after incubation in the 1 ⁇ SCPS or 4.5 ⁇ SCPS for 48 h, and FIG. 4 B was a calcium-phosphorus ratio calculated by analyzing data using EDS.
  • the coatings formed after the titanium discs of 1 ⁇ SCPS and 4.5 ⁇ SCPS groups mainly consisted of calcium, phosphorus, and oxygen elements after 48 h of incubation.
  • Ca/P ratios of the coatings also showed a similar change law: the Ca/P ratio firstly decreased and then increased followed within 5 h and the final Ca/P ratios of the 1 ⁇ SCPS and 4.5 ⁇ SCPS were 1.35 and 1.26 respectively.
  • FIG. 5 showed attenuated total reflection-fourier transform infrared spectroscopy (ATR-FTIR) analysis spectrums to analyze chemical compositions of CaP coatings after incubation of the titanium discs containing amorphous CaP coatings in 1 ⁇ SCPS ( FIG. 5 A or 4.5 ⁇ SCPS ( FIG. 5 B for 5 h, 24 h, and 48 h.
  • ATR-FTIR attenuated total reflection-fourier transform infrared spectroscopy
  • FIGS. 5 A and 5 B the ATR-FTIR spectrums showed characteristic peaks PO 4 3 ⁇ for the 1 ⁇ SCPS and 4.5 ⁇ SCPS groups at wavelengths of 1,018-1,021 cm ⁇ 1 (v3) and 560 cm ⁇ 1 (v4), indicating that the biomimetic CaP coating was successfully deposited onto the titanium disc.
  • FIGS. 6 A and 6 B showed x-ray diffraction (XRD) analysis patterns analyzing phase compositions of CaP coatings after incubation of titanium discs containing amorphous CaP coatings in 1 ⁇ SCPS ( FIG. 6 A ) or 4.5 ⁇ SCPS ( FIG. 6 B ) for 5 h, 24 h, and 48 h.
  • the coating formed in the 4.5 ⁇ SCPS also had two characteristic peaks of the hydroxyapatite.
  • DCPD dicalcium phosphate dihydrate
  • DCPA dicalcium phosphate anhydrous
  • OCP octacalcium phosphate
  • pHAp precipitated hydroxyapatite
  • the DCPD can be used in the preparation of a CaP bone cement. Studies find that the DCPD can be converted to hydroxyapatite in vivo and can be degraded and replaced
  • a 1 ⁇ SCPS coating in the present example was prepared by using the coating prepared by the method provided in example 1, wherein a volume of 1 ⁇ SCPS for immersing a titanium disc was 6 ml, and incubation time was 48 h.
  • a 4.5 ⁇ SCPS coating was prepared by using the coating prepared by the method provided in example 2, wherein a volume of 4.5 ⁇ SCPS for immersing a titanium disc was 1.5 ml, and incubation time was 48 h.
  • an acidic solution was selected to simulate effects of a surgical site (an initial environment may be acidic) and osteoclasts (which degraded bone through an acid secretion).
  • a pH 7.4
  • a release amount of Ca 2+ was relatively low.
  • the release curves of the Ca 2+ were the same for both SCPSs with an average release of about 0.17 ⁇ g per day.
  • release profiles of both coatings remained substantially the same with an average daily release amount of 0.78 ⁇ g. This phenomenon was consistent with a melting curve of a zinc ion from a hydroxyapatite coating.
  • the total amount of the Ca 2+ released by the 1 ⁇ SCPS group (59.95 ⁇ 1.39 ⁇ g) was significantly higher than that by the 4.5 ⁇ SCPS group (32.67 ⁇ 0.03 ⁇ g) (p ⁇ 0.001).
  • the Ca 2+ release in the 4.5 ⁇ SCPS coating did not change significantly, which could be explained by its solid structure and more biomimetic CaP deposited on the titanium disc.
  • a coating thickness (73 ⁇ m) produced by 1.5 mL of 4.5 ⁇ SCPS was similar to a coating thickness produced by 6 mL of 1 ⁇ SCPS on a single titanium disc (with a length and width of 4 mm respectively and a thickness of 1 mm). Therefore, the 1 ⁇ SCPS coating in the present example was prepared by using the coating prepared by the method provided in example 1, wherein a volume of 1 ⁇ SCPS for immersing a titanium disc was 6 ml.
  • the 4.5 ⁇ SCPS coating was prepared by using the coating prepared by the method provided in example 2, wherein a volume of 4.5 ⁇ SCPS for immersing a titanium disc was 1.5 ml.
  • FITC-BSA fluorescein isothiocyanate-labeled bovine serum albumin
  • a fluorescence intensity was read with a spectrophotometer (an excitation wavelength of 485 nm and an emission wavelength of 519 nm) and a FITC-BSA standard curve was plotted to calculate the FITC-BSA content.
  • FIG. 8 showed the encapsulation efficiency of the model protein FITC-BSA in the coatings of the titanium discs in the 1 ⁇ SCPS or 4.5 ⁇ SCPS groups, where, ***, p ⁇ 0.001.
  • the encapsulation efficiency of the FITC-BSA in the 4.5 ⁇ SCPS coating was 81.20 ⁇ 6.42%, which was significantly higher than 21.86 ⁇ 1.90% (p ⁇ 0.001) of the 1 ⁇ SCPS coating.
  • the 4.5 ⁇ SCPS groups had such a high FITC-BSA encapsulation efficiency largely due to that a solution volume was reduced and the coating yield was increased.

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